Disrupting the interaction between a p53 gain-of-function mutant and the transcriptional co-activator PC4 reverses drug resistance in cancer cells.

PC4 is a chromatin-associated protein and transcriptional coactivator whose role in gene regulation by wild-type p53 is now well known. Little is known about the roles of PC4 in tumor cells bearing mutant p53 genes. We show that PC4 associates with one of the tumor-associated gain-of-function p53 mutants, R273H. This association drives its recruitment to two promoters, UBE2C and MDR1, known to be responsible for imparting aggressive growth and resistance to many drugs. Here, we introduced a peptide that disrupts the PC4-R273Hp53 interaction to tumor cells bearing the R273HTP53 gene, which led to a lowering of MDR1 expression and abrogation of drug resistance in a mutant-specific manner. The results suggest that the PC4-R273Hp53 interaction may be a promising target for reducing proliferation and drug resistance in tumors.

Activator of KAT3 histone acetyltransferase family ameliorates a neurodevelopmental disorder phenotype in the syntaxin 1A ablated mouse model.

Syntaxin-1A (stx1a) repression causes a neurodevelopmental disorder phenotype, low latent inhibition (LI) behavior, by disrupting 5-hydroxytryptaminergic (5-HTergic) systems. Herein, we discovered that lysine acetyltransferase (KAT) 3B increases stx1a neuronal transcription and TTK21, a KAT3 activator, induces stx1a transcription and 5-HT release in vitro. Furthermore, glucose-derived CSP-TTK21 could restore decreased stx1a expression, 5-HTergic systems in the brain, and low LI in stx1a (+/-) mice by crossing the blood-brain barrier, whereas the KAT3 inhibitor suppresses stx1a expression, 5-HTergic systems, and LI behaviors in wild-type mice. Finally, in wild-type and stx1a (-/-) mice treated with IKK inhibitors and CSP-TTK21, respectively, we show that KAT3 activator-induced LI improvement is a direct consequence of KAT3B-stx1a pathway, not a side effect. In conclusion, KAT3B can positively regulate stx1a transcription in neurons, and increasing neuronal stx1a expression and 5-HTergic systems by a KAT3 activator consequently improves the low LI behavior in the stx1a ablation mouse model.

The Largest Subunit of Human TFIIIC Complex, TFIIIC220, a Lysine Acetyltransferase Targets Histone H3K18.

TFIIIC is a multi-subunit complex required for tRNA transcription by RNA polymerase III. Human TFIIIC holo-complex possesses lysine acetyltransferase activity that aids in relieving chromatin-mediated repression for RNA polymerase III-mediated transcription and chromatin assembly. Here we have characterized the acetyltransferase activity of the largest and DNA-binding subunit of TFIIIC complex, TFIIIC220. Purified recombinant human TFIIIC220 acetylated core histones H3, H4 and H2A in vitro. Moreover, we have identified the putative catalytic domain of TFIIIC220 that efficiently acetylates core histones in vitro. Mutating critical residues of the putative acetyl-CoA binding 'P loop' drastically reduced the catalytic activity of the acetyltransferase domain. Further analysis showed that the knockdown of TFIIIC220 in mammalian cell lines dramatically reduces global H3K18 acetylation level, which was rescued by overexpression of the putative acetyltransferase domain of human TFIIIC220. Our findings indicated a possibility of a crucial role for TFIIIC220 in maintaining acetylation homeostasis in the cell.

Delivery of chemotherapeutic drug targeting folate receptor to oral cancer cells using functionalized carbon nanospheres.

Folate receptor (FR) (α) has long been the subject of active interest as regards its potential to serve as a target for cancer therapy. FR has been found to be overexpressed in several cancers, including clinical samples of different stages from OSCC (oral squamous cell carcinoma) patients. However, no clear correlation or conclusive finding has been obtained so far which might indicate the efficacy of FR as a credible target for the treatment of OSCC. All cell lines to be used were assessed for FR-expression. Subsequently, we developed glucose-derived carbon nanospheres (CSPs) and primed them with a Folate-based cationic lipid FA8 and the chemotherapeutic drug doxorubicin (DOX). CSP based delivery systems along with pristine drug DOX were characterized and treated subsequently toin vitrocultures of OSCC cells and assessed for cancer cell targetability as well as cell death. Subsequently, treatment was administered to immunocompetent C57 mice carrying MOC2 based syngeneic OSCC tumours and assessed for tumour regression and toxicity. Ligand primed targeted CSPs exhibited commendable drug uptake as well as efficient induction of cell death. Further, receptor blocking studies revealed FR-mediated uptake, preferentially in cancer cells. Drug once delivered by ligand-primed CSPs was retained longer inside cells than pristine drug alone, indicating possibilities of better therapeutic outcome. In animal studies, CSP-FA8-DOX (Ligand primed targeted CSP) demonstrated significant regression in tumour size compared to pristine DOX as well as CSP-DOX (non-targeted CSP) treated animals. FR-mediated system CFD demonstrated targeted drug uptake and apoptotic death selectively in cancer cells. Significant tumour regression was also observedin vivo. Overall, it may be presumed that the FR is a therapeutic target with substantial potential in OSCC treatment.

Lysine Acetyltransferases (KATs) in Disguise: Diseases Implications.

Acetylation is one of the key post-translational protein modifications catalysed by the protein lysine acetyltransferases (KATs). KATs catalyse the transfer of acetyl groups to the epsilon-amino groups of lysine residues in histones and non-histone proteins. Because of its wide range of target proteins, KATs regulate many biological processes, and their aberrant activities may underlie several human diseases, including cancer, asthma, Chronic Obstructive Pulmonary Disease (COPD), and neurological disorders. Unlike most of the histone modifying enzymes, such as lysine methyltransferases, KATs do not possess any conserved domain like SET domain of lysine methyltransferases. However, almost all the major families of KATs are found to be transcriptional coactivators or adaptor proteins, with defined catalytic domains, called canonical KATs. Over the past two decades, a few proteins have been discovered to possess intrinsic KAT activity but are not classical coactivators. We would like to categorize them as non-canonical KATs (NC-KATs). These NC-KATs include general transcription factors TAFII250, mammalian TFIIIC complex, and mitochondrial protein GCN5L1, etc. This review focuses on our understanding, as well as controversies regarding non-canonical KATs, where we compare the structural and functional similarities and dissimilarities of non-canonical KATs with the canonical KATs. This review also highlights the potential role of NC-KATs in health and diseases.

Molecular determinants associated with temporal succession of SARS-CoV-2 variants in Uttar Pradesh, India.

The emergence and rapid evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused a global crisis that required a detailed characterization of the dynamics of mutational pattern of the viral genome for comprehending its epidemiology, pathogenesis and containment. We investigated the molecular evolution of the SASR-CoV-2 genome during the first, second and third waves of COVID-19 in Uttar Pradesh, India. Nanopore sequencing of the SARS-CoV-2 genome was undertaken in 544 confirmed cases of COVID-19, which included vaccinated and unvaccinated individuals. In the first wave (unvaccinated population), the 20A clade (56.32%) was superior that was replaced by 21A Delta in the second wave, which was more often seen in vaccinated individuals in comparison to unvaccinated (75.84% versus 16.17%, respectively). Subsequently, 21A delta got outcompeted by Omicron (71.8%), especially the 21L variant, in the third wave. We noticed that Q677H appeared in 20A Alpha and stayed up to Delta, D614G appeared in 20A Alpha and stayed in Delta and Omicron variants (got fixed), and several other mutations appeared in Delta and stayed in Omicron. A cross-sectional analysis of the vaccinated and unvaccinated individuals during the second wave revealed signature combinations of E156G, F157Del, L452R, T478K, D614G mutations in the Spike protein that might have facilitated vaccination breach in India. Interestingly, some of these mutation combinations were carried forward from Delta to Omicron. In silico protein docking showed that Omicron had a higher binding affinity with the host ACE2 receptor, resulting in enhanced infectivity of Omicron over the Delta variant. This work has identified the combinations of key mutations causing vaccination breach in India and provided insights into the change of [virus's] binding affinity with evolution, resulting in more virulence in Delta and more infectivity in Omicron variants of SARS-CoV-2. Our findings will help in understanding the COVID-19 disease biology and guide further surveillance of the SARS-CoV-2 genome to facilitate the development of vaccines with better efficacies.

Catecholase-catalyzed synthesis of wedelolactone, a natural coumestan and its analogs.

Biocatalysis plays an important role in the synthesis of complex organic molecules. Wedelolactone, a natural coumestan, has been reported to have many bioactive properties. A novel and efficient enzyme obtained from sweet potato juice was used for condensation of 4-hydroxycoumarins with catechols to produce wedelolactone and its structurally diverse analogs in moderate to good yields under mild reaction conditions. Hence, this enzymatic approach creates an opportunity to access many coumestan-based compounds that are potential building blocks for the synthesis of pharmaceutically important molecules.

Autophagy in Cancer: A Metabolic Perspective.

Autophagy is an intracellular catabolic degradative process in which damaged cellular organelles, unwanted proteins and different cytoplasmic components get recycled to maintain cellular homeostasis or metabolic balance. During autophagy, a double membrane vesicle is formed to engulf these cytosolic materials and fuse to lysosomes wherein the entire cargo degrades to be used again. Because of this unique recycling ability of cells, autophagy is a universal stress response mechanism. Dysregulation of autophagy leads to several diseases, including cancer, neurodegeneration and microbial infection. Thus, autophagy machineries have become targets for therapeutics. This chapter provides an overview of the paradoxical role of autophagy in tumorigenesis in the perspective of metabolism.

Metabolic Regulation of Lysine Acetylation: Implications in Cancer.

Lysine acetylation is the second most well-studied post-translational modification after phosphorylation. While phosphorylation regulates signaling cascades, one of the most significant roles of acetylation is regulation of chromatin structure. Acetyl-coenzyme A (acetyl-CoA) serves as the acetyl group donor for acetylation reactions mediated by lysine acetyltransferases (KATs). On the other hand, NAD+ serves as the cofactor for lysine deacetylases (KDACs). Both acetyl-CoA and NAD+ are metabolites integral to energy metabolism, and therefore, their metabolic flux can regulate the activity of KATs and KDACs impacting the epigenome. In this chapter, we review our current understanding of how metabolic pathways regulate lysine acetylation in normal and cancer cells.

Butyrylation Meets Adipogenesis-Probed by a p300-Catalyzed Acylation-Specific Small Molecule Inhibitor: Implication in Anti-obesity Therapy.

The enzyme p300, besides having acetyltransferase activity, can also catalyze other acylation modifications, whose physiological implications are still being investigated. Here, we report that the level of histone butyrylation increases globally as well as locally in the promoters of pro-adipogenic genes during adipogenesis. To delineate the role of p300-catalyzed butyrylation from acetylation in adipogenesis, we identified a semisynthetic derivative (LTK-14A) of garcinol, which specifically inhibited histone butyrylation without affecting acetylation. Treatment of 3T3L1 cells with LTK-14A abolished adipogenesis with downregulation of pro-adipogenic genes along with inhibition of H4K5 butyrylation. Administering LTK-14A to high-fat diet-fed and genetically obese db/db mice led to attenuation/decrease in their weight gain. The reduced obesity could be partially attributed to the inhibition of H4K5 butyrylation in adipocytes and liver. This report therefore not only, for the first time, causally links histone butyrylation with adipogenesis but also presents a probable candidate for anti-obesity therapeutics.

CBP/p300 activation promotes axon growth, sprouting, and synaptic plasticity in chronic experimental spinal cord injury with severe disability.

The interruption of spinal circuitry following spinal cord injury (SCI) disrupts neural activity and is followed by a failure to mount an effective regenerative response resulting in permanent neurological disability. Functional recovery requires the enhancement of axonal and synaptic plasticity of spared as well as injured fibres, which need to sprout and/or regenerate to form new connections. Here, we have investigated whether the epigenetic stimulation of the regenerative gene expression program can overcome the current inability to promote neurological recovery in chronic SCI with severe disability. We delivered the CBP/p300 activator CSP-TTK21 or vehicle CSP weekly between week 12 and 22 following a transection model of SCI in mice housed in an enriched environment. Data analysis showed that CSP-TTK21 enhanced classical regenerative signalling in dorsal root ganglia sensory but not cortical motor neurons, stimulated motor and sensory axon growth, sprouting, and synaptic plasticity, but failed to promote neurological sensorimotor recovery. This work provides direct evidence that clinically suitable pharmacological CBP/p300 activation can promote the expression of regeneration-associated genes and axonal growth in a chronic SCI with severe neurological disability.

Glucose derived carbon nanosphere (CSP) conjugated TTK21, an activator of the histone acetyltransferases CBP/p300, ameliorates amyloid-beta 1-42 induced deficits in plasticity and associativity in hippocampal CA1 pyramidal neurons.

The master epigenetic regulator lysine acetyltransferase (KAT) p300/CBP plays a pivotal role in neuroplasticity and cognitive functions. Recent evidence has shown that in several neurodegenerative diseases, including Alzheimer's disease (AD), the expression level and function of p300/CBP are severely compromised, leading to altered gene expression causing pathological conditions. Here, we show that p300/CBP activation by a small-molecule TTK21, conjugated to carbon nanosphere (CSP) ameliorates Aß-impaired long-term potentiation (LTP) induced by high-frequency stimulation, theta burst stimulation, and synaptic tagging/capture (STC). This functional rescue was correlated with CSP-TTK21-induced changes in transcription and translation. Mechanistically, we observed that the expression of a large number of synaptic plasticity- and memory-related genes was rescued, presumably by the restoration of p300/CBP mediated acetylation. Collectively, these results suggest that small-molecule activators of p300/CBP could be a potential therapeutic molecule for neurodegenerative diseases like AD.

Phosphorylation-dependent association of human chromatin protein PC4 to linker histone H1 regulates genome organization and transcription.

Human Positive Coactivator 4 (PC4) is a multifaceted chromatin protein involved in diverse cellular processes including genome organization, transcription regulation, replication, DNA repair and autophagy. PC4 exists as a phospho-protein in cells which impinges on its acetylation by p300 and thereby affects its transcriptional co-activator functions via double-stranded DNA binding. Despite the inhibitory effects, the abundance of phosphorylated PC4 in cells intrigued us to investigate its role in chromatin functions in a basal state of the cell. We found that casein kinase-II (CKII)-mediated phosphorylation of PC4 is critical for its interaction with linker histone H1. By employing analytical ultracentrifugation and electron microscopy imaging of in vitro reconstituted nucleosomal array, we observed that phospho-mimic (PM) PC4 displays a superior chromatin condensation potential in conjunction with linker histone H1. ATAC-sequencing further unveiled the role of PC4 phosphorylation to be critical in inducing chromatin compaction of a wide array of coding and non-coding genes in vivo. Concordantly, phospho-PC4 mediated changes in chromatin accessibility led to gene repression and affected global histone modifications. We propose that the abundance of PC4 in its phosphorylated state contributes to genome compaction contrary to its co-activator function in driving several cellular processes like gene transcription and autophagy.

vp1524, a Vibrio parahaemolyticus NAD +-dependent deacetylase, regulates host response during infection by induction of host histone deacetylation.

Gram-negative intracellular pathogen Vibrio parahaemolyticus manifests its infection through a series of effector proteins released into the host via the type III secretion system. Most of these effector proteins alter signalling pathways of the host to facilitate survival and proliferation of bacteria inside host cells. Here, we report V. parahaemolyticus (serotype O3:K6) infection-induced histone deacetylation in host intestinal epithelial cells, particularly deacetylation of H3K9, H3K56, H3K18 and H4K16 residues. We found a putative NAD+-dependent deacetylase, vp1524 (vpCobB) of V. parahaemolyticus, was overexpressed during infection. Biochemical assays revealed that Vp1524 is a functional NAD+-dependent Sir2 family deacetylase in vitro, which was capable of deacetylating acetylated histones. Furthermore, we observed that vp1524 is expressed and localized to the nuclear periphery of the host cells during infection. Consequently, Vp1524 translocated to nuclear compartments of transfected cells, deacetylated histones, specifically causing deacetylation of those residues (K56, K16, K18) associated with V. parahaemolyticus infection. This infection induced deacetylation resulted in transcriptional repression of several host genes involved in epigenetic regulation, immune response, autophagy etc. Thus, our study shows that a V. parahaemolyticus lysine deacetylase Vp1524 is secreted inside the host cells during infection, modulating host gene expression through histone deacetylation.

Visible-light excited polar Dion-Jacobson Rb(Bi1-xEux)2Ti2NbO10 perovskites: photoluminescence properties and in vitro bioimaging.

Rare-earth ion-activated oxide phosphors are beneficial to overcome problems like photobleaching, reduced lifetime, and the blinking of organic dyes and quantum dots for bioimaging applications. In this work, we report that the phosphors Rb(Bi1-xEux)2Ti2NbO10 (0.025 ≤ x ≤ 0.2) exhibit an electric dipole moment induced sharp 5D0 → 7F2 transition upon blue light excitation with a luminescence lifetime of ∼1 ms. While the major drawback of Eu3+ activated compounds is the requirement of harmful UV excitation, interestingly, this solid solution exhibits a sharp and intense excitation peak at 465 nm (visible light) compared to 363 and 395 nm (UV region), making it viable for bioimaging applications. The sample with x = 0.125 reveals the highest emission intensity with a quantum yield of 10.5%. Temperature-dependent emission spectra of the sample (x = 0.125) reveal excellent thermal stability. The low cytotoxicity of this compound is confirmed by incubation in HeLa cells and SH-SY5Y neuroblastoma cells. The biocompatibility of the compound with SH-SY5Y and HEK293 cells was imaged via two-photon microscopy, indicating its potential for biomedical applications.

Histone Chaperone Nucleophosmin Regulates Transcription of Key Genes Involved in Oral Tumorigenesis.

Nucleophosmin (NPM1) is a multifunctional histone chaperone that can activate acetylation-dependent transcription from chromatin templates in vitro. p300-mediated acetylation of NPM1 has been shown to further enhance its transcription activation potential. Acetylated and total NPM1 pools are increased in oral squamous cell carcinoma. However, the role of NPM1 or its acetylated form (AcNPM1) in transcriptional regulation in cells and oral tumorigenesis is not fully elucidated. Using ChIP-seq analyses, we provide the first genome-wide profile of AcNPM1 and show that AcNPM1 is enriched at transcriptional regulatory elements. AcNPM1 co-occupies marks of active transcription at promoters and DNase I hypersensitive sites at enhancers. In addition, using a high-throughput protein interaction profiling approach, we show that NPM1 interacts with RNA Pol II, general transcription factors, mediator subunits, histone acetyltransferase complexes, and chromatin remodelers. NPM1 histone chaperone activity also contributes to its transcription activation potential. Further, NPM1 depletion leads to decreased AcNPM1 occupancy and reduced expression of genes required for proliferative, migratory and invasive potential of oral cancer cells. NPM1 depletion also abrogates the growth of orthotopic tumors in mice. Collectively, these results establish that AcNPM1 functions as a coactivator during during RNA polymerase II-driven transcription and regulates the expression of genes that promote oral tumorigenesis.

Phase III, Randomized, Double-blind, Placebo controlled trial of Efficacy, Safety and Tolerability of Antiviral drug Umifenovir vs Standard care of therapy in non-severe COVID-19 patients.

OBJECTIVE: To test efficacy, safety and tolerability of Umifenovir in non-severe COVID-19 adult patients. METHODS: We carried out randomized, double-blind, placebo-controlled, multicenter, phase III trials involving adult (18-75 years), non-severe COVID19 patients, randomized 1:1 on placebo or Umifenovir (800 mg BID, maximum 14 days) respectively along with standard-of-care. The primary endpoint for Asymptotic-mild patients was time to nasopharyngeal swab RT-PCR test negativity. For Moderate patients, the average change in the ordinal scale from the baseline scores on the eight-point WHO ordinal scale was assessed. RESULTS: 132 patients were recruited between 3rd October to 28th April 2021, of which 9 discontinued due to various reasons. In Mild-asymptomatic patients (n=82), we found that 73% patients in the Umifenovir arm were RT-PCR negative, while 40% patients in the placebo arm were negative (P=0.004) on day 5. However, in the moderate group (n=41), the WHO scores for the Umifenovir arm was not statistically significant (P=0.125 on day 3), while it was statistically significant in the Mild-asymptomatic group (P=0.019 on day 5). CONCLUSION: Umifenovir meets the primary and secondary endpoint criteria and exhibits statistically significant efficacy for Mild-asymptomatic patients. It is efficacious, safe and well-tolerated at the tested dosage of 800mg BID, maximum 14 days.

14-3-3γ prevents centrosome duplication by inhibiting NPM1 function.

14-3-3 proteins bind to ligands via phospho-serine containing consensus motifs. However, the molecular mechanisms underlying complex formation and dissociation between 14-3-3 proteins and their ligands remain unclear. We identified two conserved acidic residues in the 14-3-3 peptide-binding pocket (D129 and E136) that potentially regulate complex formation and dissociation. Altering these residues to alanine led to opposing effects on centrosome duplication. D129A inhibited centrosome duplication, whereas E136A stimulated centrosome amplification. These results were due to the differing abilities of these mutant proteins to form a complex with NPM1. Inhibiting complex formation between NPM1 and 14-3-3γ led to an increase in centrosome duplication and over-rode the ability of D129A to inhibit centrosome duplication. We identify a novel role of 14-3-3γ in regulating centrosome licensing and a novel mechanism underlying the formation and dissociation of 14-3-3 ligand complexes dictated by conserved residues in the 14-3-3 family.

Chromatin Protein PC4 Orchestrates B Cell Differentiation by Collaborating with IKAROS and IRF4.

The chromatin protein positive coactivator 4 (PC4) has multiple functions, including chromatin compaction. However, its role in immune cells is largely unknown. We show that PC4 orchestrates chromatin structure and gene expression in mature B cells. B-cell-specific PC4-deficient mice show impaired production of antibody upon antigen stimulation. The PC4 complex purified from B cells contains the transcription factors (TFs) IKAROS and IRF4. IKAROS protein is reduced in PC4-deficient mature B cells, resulting in de-repression of their target genes in part by diminished interactions with gene-silencing components. Upon activation, the amount of IRF4 protein is not increased in PC4-deficient B cells, resulting in reduction of plasma cells. Importantly, IRF4 reciprocally induces PC4 expression via a super-enhancer. PC4 knockdown in human B cell lymphoma and myeloma cells reduces IKAROS protein as an anticancer drug, lenalidomide. Our findings establish PC4 as a chromatin regulator of B cells and a possible therapeutic target adjoining IKAROS in B cell malignancies.